The genome of vesicular stomatitis virus is single stranded RNA which is complementary to its messenger RNA. The transcription of the genome is mediated by an encapsidated transcriptase, and has been studied extensively. On the other hand, the mechanisms by which the genome RNA is replicated is not understood. The two processes are distinct, but intimately linked. The identity and function of virus specific proteins involved in these pathways and the nature of their interactions will be studied by an analysis of temperature sensitive mutants which display defective RNA synthesis. Temperature sensitive mutants of the New Jersey serotype will be employed since they provide the maximum number of complementation groups affecting RNA synthesis. The analysis will comprise an integration of two basic approaches: 1) chemical analysis to identify the altered protein; 2) refined functional analysis at the level of the individual protein subunits. In addition the temperature sensitive mutants in conjunction with defective interfering particles will be exploited in attempts to develop a system for isolating the replication pathway in vivo and in vitro.